Sustainable strategies: Nature-based solutions to tackle antibiotic resistance gene proliferation and improve agricultural productivity and soil quality.

The issue of antibiotic resistance is now recognized by the World Health Organisation (WHO) as one of the major problems in human health. Although its effects are evident in the healthcare settings, the root cause should be traced back to the One Health link, extending from animals to the environment. In fact, the use of organic fertilizers in agroecosystems represents one, if not the primary, cause of the introduction of antibiotics and antibiotic-resistant bacteria into the soil. Since the concentrations of antibiotics introduced into the soil are residual, the agroecosystem has become a perfect environment for the selection and proliferation of antibiotic resistance genes (ARGs). The continuous influx of these emerging contaminants (i.e., antibiotics) into the agroecosystem results in the selection and accumulation of ARGs in soil bacteria, occasionally giving rise to multi-resistant bacteria. These bacteria may harbour ARGs related to various antibiotics on their plasmids. In this context, these bacteria can potentially enter the human sphere when individuals consume food from contaminated agroecosystems, leading to the acquisition of multi-resistant bacteria. Once introduced into the nosocomial environment, these bacteria pose a significant threat to human health. In this review, we analyse how the use of digestate as an organic fertilizer can mitigate the spread of ARGs in agroecosystems. Furthermore, we highlight how, according to European guidelines, digestate can be considered a Nature-Based Solution (NBS). This NBS not only has the ability to mitigate the spread of ARGs in agroecosystems but also offers the opportunity to further improve Microbial-Based Solutions (MBS), with the aim of enhancing soil quality and productivity.

Lactiplantibacillus plantarum monolayer enhanced bactericidal action of carvacrol: biofilm inhibition of viable foodborne pathogens and spoilage microorganisms.

The prevalence of biofilm-associated microorganisms and the increasing use of ready-to-eat fresh products represent the current duality the food industry must address. Innovative and eco-friendly antibiofilm solutions and appropriate microbiological food control systems are urgently needed to improve food quality and safety. This study aimed to investigate the in vitro combined efficacy of carvacrol with a pre-formed biofilm monolayer of the probiotic Lactiplantibacillus plantarum DSM 20174. The antimicrobial activity of carvacrol against both planktonic and sessile cells of foodborne pathogens and spoilage microorganisms, alone or in the presence of the pre-formed biofilm of L. plantarum, was investigated by culture-based methods along with flow cytometry (FCM) to monitor cells' cultivability and viability. The synergistic action of carvacrol and the pre-formed biofilm of L. plantarum was evaluated in the 96-well plates. The results showed that L. plantarum pre-formed biofilm monolayer enhanced the antimicrobial effect of carvacrol determining a bactericidal action while the treatment alone induced the viable but not culturable (VBNC) cell state only. Furthermore, the great efficacy of the combined treatment allowed the application of a lower concentration of carvacrol (100 ppm) to achieve significant damage in cell viability. In conclusion, the incorporation of carvacrol into the L. plantarum pre-formed biofilm represents a promising alternative for an antimicrobial functionalized ready-to-eat packaging.

A Metagenomic and Gene Expression Analysis in Wheat (T. durum) and Maize (Z. mays) Biofertilized with PGPM and Biochar.

Commodity crops, such as wheat and maize, are extremely dependent on chemical fertilizers, a practice contributing greatly to the increase in the contaminants in soil and water. Promising solutions are biofertilizers, i.e., microbial biostimulants that when supplemented with soil stimulate plant growth and production. Moreover, the biofertilizers can be fortified when (i) provided as multifunctional consortia and (ii) combined with biochar with a high cargo capacity. The aim of this work was to determine the molecular effects on the soil microbiome of different biofertilizers and delivery systems, highlight their physiological effects and merge the data with statistical analyses. The measurements of the physiological parameters (i.e., shoot and root biomass), transcriptomic response of genes involved in essential pathways, and characterization of the rhizosphere population were analyzed. The results demonstrated that wheat and maize supplemented with different combinations of selected microbial consortia and biochar have a positive effect on plant growth in terms of shoot and root biomass; the treatments also had a beneficial influence on the biodiversity of the indigenous rhizo-microbial community, reinforcing the connection between microbes and plants without further spreading contaminants. There was also evidence at the transcriptional level of crosstalk between microbiota and plants.

Synergistic Action of Mild Heat and Essential Oil Treatments on Culturability and Viability of Escherichia coli ATCC 25922 Tested In Vitro and in Fruit Juice.

The strengthening effect of a mild temperature treatment on the antimicrobial efficacy of essential oils has been widely reported, often leading to an underestimation or a misinterpretation of the product's microbial status. In the present study, both a traditional culture-based method and Flow Cytometry (FCM) were applied to monitor the individual or combined effect of Origanum vulgare essential oil (OEO) and mild heat treatment on the culturability and viability of Escherichia coli in a conventional culture medium and in a fruit juice challenge test. The results obtained in the culture medium showed bacterial inactivation with an increasing treatment temperature (55 °C, 60 °C, 65 °C), highlighting an overestimation of the dead population using the culture-based method; in fact, when the FCM method was applied, the prevalence of injured bacterial cells in a viable but non-culturable (VBNC) state was observed. When commercial fruit juice with a pH of 3.8 and buffered at pH 7.0 was inoculated with E. coli ATCC 25922, a bactericidal action of OEO and a higher efficiency of the mild heat at 65 °C for 5' combined with OEO were found. Overall, the combination of mild heat and OEO treatment represents a promising antimicrobial alternative to improve the safety of fruit juice.

Development of a predictive model of the microbial inactivation of L. monocytogenes during low thermal treatment of fruit juices in combination with carvacrol as aroma compound.

Mild heat treatment of fruit juices in combination with natural aroma compounds has been reported as an alternative to conventional pasteurization to better preserve their nutritional value. However, its antimicrobial efficiency varies from one juice to another. This study aims at developing a secondary predictive model of microbial inactivation scale during such combined process. Carvacrol was used as aroma compound and acid-adapted L. monocytogenes as target microorganism. The inactivation kinetics of this bacteria were followed in simulated fruit juices using a Central Composite Design with pH (2-6), °Brix (0-24), temperature (55-65 °C), and carvacrol concentration (0-60 µL/L) as independent variables. Curves were fitted to the Weibull inactivation model, and data collected used to generate two predictive models of the inactivation scale parameter through multiple regression analysis following an empirical and a mechanistic (based on Gamma concept) approach. The best of the two models was then validated using real fruit (orange, pineapple, and watermelon) juices. The empirical model where only the four variables tested were considered showed a lower statistical performance compared to the mechanistic model where octanol-water partition coefficient (Ko/w) and vapour pressure (Vp) of carvacrol at the treatment temperature were integrated (R2 0.6 and 0.9; Accuracy factor 1.5 and 1.3; Sum of Squared Error 3.6 and 1.1, respectively). No significant difference was observed between inactivation scale values obtained with real juices and the predicted values calculated using this mechanistic model. The Ko/w and Vp of the aroma compound used are key parameters that determine the efficiency of the above-described combined treatment.

Lung and Gut Microbiota Changes Associated with Pseudomonas aeruginosa Infection in Mouse Models of Cystic Fibrosis.

Cystic fibrosis (CF) disease leads to altered lung and gut microbiomes compared to healthy subjects. The magnitude of this dysbiosis is influenced by organ-specific microenvironmental conditions at different stages of the disease. However, how this gut-lung dysbiosis is influenced by Pseudomonas aeruginosa chronic infection is unclear. To test the relationship between CFTR dysfunction and gut-lung microbiome under chronic infection, we established a model of P. aeruginosa infection in wild-type (WT) and gut-corrected CF mice. Using 16S ribosomal RNA gene, we compared lung, stool, and gut microbiota of C57Bl/6 Cftr tm1UNCTgN(FABPCFTR) or WT mice at the naïve state or infected with P. aeruginosa. P. aeruginosa infection influences murine health significantly changing body weight both in CF and WT mice. Both stool and gut microbiota revealed significantly higher values of alpha diversity in WT mice than in CF mice, while lung microbiota showed similar values. Infection with P. aeruginosa did not changed the diversity of the stool and gut microbiota, while a drop of diversity of the lung microbiota was observed compared to non-infected mice. However, the taxonomic composition of gut microbiota was shown to be influenced by P. aeruginosa infection in CF mice but not in WT mice. This finding indicates that P. aeruginosa chronic infection has a major impact on microbiota diversity and composition in the lung. In the gut, CFTR genotype and P. aeruginosa infection affected the overall diversity and taxonomic microbiota composition, respectively. Overall, our results suggest a cross-talk between lung and gut microbiota in relation to P. aeruginosa chronic infection and CFTR mutation.

Identification of Beneficial Microbial Consortia and Bioactive Compounds with Potential as Plant Biostimulants for a Sustainable Agriculture.

A growing body of evidence demonstrates the potential of various microbes to enhance plant productivity in cropping systems although their successful field application may be impaired by several biotic and abiotic constraints. In the present work, we aimed at developing multifunctional synthetic microbial consortia to be used in combination with suitable bioactive compounds for improving crop yield and quality. Plant growth-promoting microorganisms (PGPMs) with different functional attributes were identified by a bottom-up approach. A comprehensive literature survey on PGPMs associated with maize, wheat, potato and tomato, and on commercial formulations, was conducted by examining peer-reviewed scientific publications and results from relevant European projects. Metagenome fragment recruitments on genomes of potential PGPMs represented in databases were also performed to help identify plant growth-promoting (PGP) strains. Following evidence of their ability to coexist, isolated PGPMs were synthetically assembled into three different microbial consortia. Additionally, the effects of bioactive compounds on the growth of individually PGPMs were tested in starvation conditions. The different combination products based on microbial and non-microbial biostimulants (BS) appear worth considering for greenhouse and open field trials to select those potentially adoptable in sustainable agriculture.

Phenotyping of Different Italian Durum Wheat Varieties in Early Growth Stage With the Addition of Pure or Digestate-Activated Biochars.

This study aims to highlight the major effects of biochar incorporation into potting soil substrate on plant growth and performance in early growth stages of five elite Italian varieties of durum wheat (Triticum durum). The biochars used were obtained from two contrasting feedstocks, namely wood chips and wheat straw, by gasification under high temperature conditions, and were applied in a greenhouse experiment either as pure or as nutrient-activated biochar obtained by incubation with digestate. The results of the experiment showed that specific genotypes as well as different treatments with biochar have significant effects on plant response when looking at shoot traits related to growth. The evaluated genotypes could be clustered in two main distinct groups presenting, respectively, significantly increasing (Duilio, Iride, and Saragolla varieties) and decreasing (Marco Aurelio and Grecale varieties) values of projected shoot system area (PSSA), fresh weight (FW), dry weight (DW), and plant water loss by evapotranspiration (ET). All these traits were correlated with Pearson correlation coefficients ranging from 0.74 to 0.98. Concerning the treatment effect, a significant alteration of the mentioned plant traits was observed when applying biochar from wheat straw, characterized by very high electrical conductivity (EC), resulting in a reduction of 34.6% PSSA, 43.2% FW, 66.9% DW, and 36.0% ET, when compared to the control. Interestingly, the application of the same biochar after nutrient spiking with digestate determined about a 15-30% relief from the abovementioned reduction induced by the application of the sole pure wheat straw biochar. Our results reinforce the current basic knowledge available on biological soil amendments as biochar and digestate.

Untargeted Metagenomic Investigation of the Airway Microbiome of Cystic Fibrosis Patients with Moderate-Severe Lung Disease.

Although the cystic fibrosis (CF) lung microbiota has been characterized in several studies, little is still known about the temporal changes occurring at the whole microbiome level using untargeted metagenomic analysis. The aim of this study was to investigate the taxonomic and functional temporal dynamics of the lower airway microbiome in a cohort of CF patients. Multiple sputum samples were collected over 15 months from 22 patients with advanced lung disease regularly attending three Italian CF Centers, given a total of 79 samples. DNA extracted from samples was subjected to shotgun metagenomic sequencing allowing both strain-level taxonomic profiling and assessment of the functional metagenomic repertoire. High inter-patient taxonomic heterogeneity was found with short-term compositional changes across clinical status. Each patient exhibited distinct sputum microbial communities at the taxonomic level, and strain-specific colonization of both traditional and atypical CF pathogens. A large core set of genes, including antibiotic resistance genes, were shared across patients despite observed differences in clinical status, and consistently detected in the lung microbiome of all subjects independently from known antibiotic exposure. In conclusion, an overall stability in the microbiome-associated genes was found despite taxonomic fluctuations of the communities.

Impact of clonally-related Burkholderia contaminans strains in two patients attending an Italian cystic fibrosis centre: a case report.

BACKGROUND: Burkholderia contaminans is one of the 20 closely related bacterial of the Burkholderia cepacia complex, a group of bacteria that are ubiquitous in the environment and capable of infecting people with cystic fibrosis (CF). This species is an emerging pathogen and it has been widely isolated from CF patients in Argentina, Spain, Portugal, Australia, Canada, USA with a low prevalence in Ireland, France, Russia, Switzerland, Czech Republic, and Italy. This is the first report of B. contaminans affecting two Italian CF patients attending the same CF Centre. We correlate B. contaminans colonisation with lung function decline and co-infection with other clinically relevant CF pathogens. CASE PRESENTATION: B. contaminans was identified by Multi Locus Sequence Typing in routine sputum analysis of two Caucasian CF women homozygous for Phe508del CFTR mutation. Sequence Type 102 was detected in both strains. It is known that B. contaminans ST102 was isolated both from CF and non-CF patients, with an intercontinental spread across the world. Random Amplified Polymorphic DNA analysis revealed the genetic relatedness between the two strains. We examined their susceptibility to antimicrobial agents, comparing the latter with that recorded for other B. contaminans isolated from different countries. We also described key virulence factors possibly linked with a clinical outcome. Specifically, we attempted to correlate colonization with the incidence of acute exacerbation of symptoms and lung function decline. CONCLUSIONS: This case presentation suggests that acquisition of B. contaminans ST102 is not directly associated with a lung function decline. We retain that the presence of other CF pathogens (i.e. MRSA and Trichosporon) along with B. contaminans ST102 might have contributed to the worsening of clinical conditions in our CF patients. The circumstances leading to the establishment of B. contaminans ST102 infections are still unknown. We highlight the importance to proper detect and typing bacteria implicated in CF infection by using molecular techniques.

Deciphering the Ecology of Cystic Fibrosis Bacterial Communities: Towards Systems-Level Integration.

Despite over a decade of cystic fibrosis (CF) microbiome research, much remains to be learned about the overall composition, metabolic activities, and pathogenicity of the microbes in CF airways, limiting our understanding of the respiratory microbiome's relation to disease. Systems-level integration and modeling of host-microbiome interactions may allow us to better define the relationships between microbiological characteristics, disease status, and treatment response. In this way, modeling could pave the way for microbiome-based development of predictive models, individualized treatment plans, and novel therapeutic approaches, potentially serving as a paradigm for approaching other chronic infections. In this review, we describe the challenges facing this effort and propose research priorities for a systems biology approach to CF lung disease.

The Impact of Soil-Applied Biochars From Different Vegetal Feedstocks on Durum Wheat Plant Performance and Rhizospheric Bacterial Microbiota in Low Metal-Contaminated Soil.

Biochar shapes the soil environment and plant growth. Nevertheless, the mechanisms associated with an improved plant biomass and soil microbiome in low metal-contaminated soils are still unclear. In this study, the influence of biochar on soil physico-chemical properties, plant performance, and rhizosphere microbiota in durum wheat was investigated at the above- and belowground levels. Two kinds of biochar from different feedstocks (wood chips and wheat straw pellets) and two Italian durum wheat varieties, Duilio and Marco Aurelio, were analyzed in a greenhouse using a low-nutrient gleyic fluvisol containing a very small amount of Pb and Zn. Four different treatments were performed: soil-only control (C), soil amended with woody biochar equilibrated with nutrient solution (B1+) and non-activated (B1-), and soil amended with non-activated (B2-) wheat straw biochar. Seven weeks after seed germination, (1) the physico-chemical properties of soil, biochars, and mixtures were assessed; (2) the fresh and dry weight of aboveground plant tissues and roots and other morphometric traits were measured; and (3) metabarcoding of the 16S rRNA bacterial gene was performed on rhizosphere soil samples. The results showed that the biochar from wheat straw had stronger impact on both durum varieties, with higher electrical conductivity, higher levels of available K and Na, and a substantial increase of dissolved Na+, K+, and Cl- ions in pore water. Generally, biochar amendment decreased Zn availability for the plants. In addition, biochar improved plant growth in the early growth stage, and the more positive effect was achieved by combining wheat straw biochar with Marco Aurelio. Rhizosphere bacterial microbiota showed variation in alpha diversity only due to treatment; on the other hand, the differential analysis showed consistent variation among samples with significant effects on amplicon sequence variant (ASV) abundance due to the specific biochar treatment as well as the genotype. The pure B1-, due to its scarce nutrient content with respect to the richer types (B1+ and B2-), had a negative impact on microbiota richness. Our study highlights that an appropriate combination of biochar feedstock and crop species may lead to superior yield.

How to Process Sputum Samples and Extract Bacterial DNA for Microbiota Analysis.

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.

Belowground Microbiota and the Health of Tree Crops.

Trees are crucial for sustaining life on our planet. Forests and land devoted to tree crops do not only supply essential edible products to humans and animals, but also additional goods such as paper or wood. They also prevent soil erosion, support microbial, animal, and plant biodiversity, play key roles in nutrient and water cycling processes, and mitigate the effects of climate change acting as carbon dioxide sinks. Hence, the health of forests and tree cropping systems is of particular significance. In particular, soil/rhizosphere/root-associated microbial communities (known as microbiota) are decisive to sustain the fitness, development, and productivity of trees. These benefits rely on processes aiming to enhance nutrient assimilation efficiency (plant growth promotion) and/or to protect against a number of (a)biotic constraints. Moreover, specific members of the microbial communities associated with perennial tree crops interact with soil invertebrate food webs, underpinning many density regulation mechanisms. This review discusses belowground microbiota interactions influencing the growth of tree crops. The study of tree-(micro)organism interactions taking place at the belowground level is crucial to understand how they contribute to processes like carbon sequestration, regulation of ecosystem functioning, and nutrient cycling. A comprehensive understanding of the relationship between roots and their associate microbiota can also facilitate the design of novel sustainable approaches for the benefit of these relevant agro-ecosystems. Here, we summarize the methodological approaches to unravel the composition and function of belowground microbiota, the factors influencing their interaction with tree crops, their benefits and harms, with a focus on representative examples of Biological Control Agents (BCA) used against relevant biotic constraints of tree crops. Finally, we add some concluding remarks and suggest future perspectives concerning the microbiota-assisted management strategies to sustain tree crops.

Omics approaches on fresh-cut lettuce reveal global molecular responses to sodium hypochlorite and peracetic acid treatment.

BACKGROUND: Lettuce is a leafy vegetable that is extensively commercialized as a ready-to-eat product because of its widespread use in human nutrition as salad. It is well known that washing treatments can severely affect the quality and shelf-life of ready-to-eat vegetables. The study presented here evaluated the effect of two washing procedures on fresh-cut lettuce during storage. RESULTS: An omics approach was applied to reveal global changes at molecular level induced by peracetic acid washing in comparison with sodium hypochlorite treatment. Microbiological analyses were also performed to quantify total bacterial abundance and composition. The study revealed wide metabolic alterations induced by the two sanitizers. In particular, transcriptomic and proteomic analyses pointed out a number of transcripts and proteins differentially accumulated in response to peracetic acid washing, mainly occurring on the first day of storage. In parallel, different microbiota composition and significant reduction in total bacterial load following washing were also observed. CONCLUSION: The results provide useful information for the fresh-cut industry to select an appropriate washing procedure preserving fresh-like attributes as much as possible during storage of the end product. Molecular evidence indicated peracetic acid to be a valid alternative to sodium hypochlorite as sanitizer solution. © 2017 Society of Chemical Industry.

Environmental Burkholderia cenocepacia Strain Enhances Fitness by Serial Passages during Long-Term Chronic Airways Infection in Mice.

Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656T. Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts.

A Different Microbiome Gene Repertoire in the Airways of Cystic Fibrosis Patients with Severe Lung Disease.

In recent years, next-generation sequencing (NGS) was employed to decipher the structure and composition of the microbiota of the airways in cystic fibrosis (CF) patients. However, little is still known about the overall gene functions harbored by the resident microbial populations and which specific genes are associated with various stages of CF lung disease. In the present study, we aimed to identify the microbial gene repertoire of CF microbiota in twelve patients with severe and normal/mild lung disease by performing sputum shotgun metagenome sequencing. The abundance of metabolic pathways encoded by microbes inhabiting CF airways was reconstructed from the metagenome. We identified a set of metabolic pathways differently distributed in patients with different pulmonary function; namely, pathways related to bacterial chemotaxis and flagellar assembly, as well as genes encoding efflux-mediated antibiotic resistance mechanisms and virulence-related genes. The results indicated that the microbiome of CF patients with low pulmonary function is enriched in virulence-related genes and in genes encoding efflux-mediated antibiotic resistance mechanisms. Overall, the microbiome of severely affected adults with CF seems to encode different mechanisms for the facilitation of microbial colonization and persistence in the lung, consistent with the characteristics of multidrug-resistant microbial communities that are commonly observed in patients with severe lung disease.

Correction: Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline.

[This corrects the article DOI: 10.1371/journal.pone.0156807.].

Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline.

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.

Bacterial community and proteome analysis of fresh-cut lettuce as affected by packaging.

With the growing demand of fresh-cut vegetables, a variety of packaging films are produced specifically to improve safety and quality of the fresh vegetables over the storage period. The aim of our work was to evaluate the influence of different packaging films on the quality of fresh-cut lettuce analyzing changes in bacterial community composition and modifications at the proteome level, by means of culture-dependent/culture-independent methods and differential gel electrophoresis combined with mass spectrometry analysis. Total viable counts indicated the presence of a highly variable and complex microbial flora, around a mean value of 6.26 log10 CFU g(-1). Analysis of terminal-restriction fragment length polymorphism data indicated that bacterial communities changed with packaging films and time, showing differences in community composition and diversity indices between the commercially available package (F) and the new packages (A and C), in the first days after packaging. Also proteomic analysis revealed significant changes, involving proteins related to energy metabolism, photosynthesis, plant defense and oxidative stress processes, between F and A/C packages. In conclusion, microbiological and proteomic analysis have proved to be powerful tools to provide new insights into both the composition of leaf-associated bacterial communities and protein content of fresh-cut lettuce during the shelf-life storage process.